Association of OPRM1 rs1799971, HTR1B rs6296 and COMT rs4680 polymorphisms with clinical phenotype among women with fibromyalgia

To investigate the association between three selected pain polymorphisms and clinical, functional, sensory-related, psychophysical, psychological or cognitive variables in a sample of women with fibromyalgia (FMS). One hundred twenty-three (n = 123) women with FMS completed demographic (age, height, weight), clinical (years with pain, intensity of pain at rest and during daily living activities), functional (quality of life, physical function), sensory-related (sensitization-associated and neuropathic-associated symptoms), psychophysical (pressure pain thresholds), psychological (sleep quality, depressive and anxiety level) and cognitive (pain catastrophizing, kinesiophobia) variables. Those three genotypes of the OPRM1 rs1799971, HTR1B rs6296 and COMT rs4680 single nucleotide polymorphisms were obtained by polymerase chain reactions from no-stimulated whole saliva collection. No significant differences in demographic, clinical, functional, sensory-related, psychophysical, psychological and cognitive variables according to OPRM1 rs1799971, HTR1B rs6296 or COMT rs4680 genotype were identified in our sample of women with FMS. A multilevel analysis did not either reveal any significant gene-to-gene interaction between OPRM1 rs1799971 x HTR1B rs6296, OPRM1 rs1799971 x COMT rs4680 and HTR1B rs6296 x COMT rs4680 for any of the investigated outcomes. This study revealed that three single nucleotide polymorphisms, OPRM1 rs1799971, HTR1B rs6296 or COMT rs4680, mostly associated with chronic pain were not involved in phenotyping features of FMS. Potential gene-to-gene interaction and their association with clinical phenotype in women with FMS should be further investigated in future studies including large sample sizes.


Patient-reported outcome measures (PROMs)
Different PROMs including clinical, functional, sensory-related, cognitive, and psychological variables were collected in a structured questionnaire created for this study.

Clinical variables
The mean intensity of pain at rest, the worst level of pain at rest, and intensity of pain experienced during daily living activities were assessed in a 10-point (0: no pain, 10: maximum pain) Numerical Pain Rate Scale (NPRS) 31 .

Functional variables
The validated version of the Fibromyalgia Impact Questionnaire (FIQ, score: 0-100 points) in Spanish language, a specific-disease PROM for FMS, was used to assess pain-related disability 32 .The FIQ consists of 10 items (from www.nature.com/scientificreports/0 to 10 points) assessing the function on daily tasks, the number of days feeling good during the previous 7 days, pain interference at work, intensity of pain, fatigue, night resting, presence of stiffness, anxiety and depressive levels.Higher scores represent greater related-disability and pain severity 32 .
The paper-based five-level version of EQ-5D-5L questionnaire, a generic PROM consisting of five dimensions evaluated from 1 (no problem) to 5 (severe problems), was used to determine health-related quality of life 33 .Responses from the EQ-5D-5L are converted into a single index number between 0 (health state judged to be equivalent to death) and 1 (optimal health status) by applying crosswalk index values for the Spanish population 33 .

Sensory-related variables
The presence of sensitization-associated symptoms was assessed with the Spanish version of the Central Sensitization Inventory (CSI) 34 .This PROM uses 5-points Likert scale responses to identify the presence of sensitizationrelated symptoms.The cut-off for determining the presence of altered nociceptive pain processing has been set at 40 points out of total score of 100 points 35 .
The presence of neuropathic-associated pain symptomatology was assessed with the Spanish version of the Self-Report Leeds Assessment of Neuropathic Symptoms (S-LANSS) questionnaire 36 .This PROM identifies with acceptable sensitivity, internal consistency and validity whether a patient experiences pain symptom to be considered predominantly or non-predominantly of neuropathic origin 36 .From a total score of 24 points, scores higher than 12 points suggest the presence of neuropathic symptoms 36 .

Psychological variables
The Hospital Anxiety and Depression Scale (HADS) was used to evaluate the levels of anxiety (HADS-A, 7-items) and depression (HADS-D, 7-items) in our sample of FMS 37 .A score of 12 points out of 21 in the HADS-A and a score of 10 points out of 21 in the HADS-D suggests the presence of anxiety and depressive levels, respectively 37 .
The quality of sleep was assessed with the Pittsburgh Sleep Quality Index (PSQI) 38 .This PROM uses 4-point Likert-type scale responses evaluating different aspects of sleep, where 8 points out of a total score of 21 suggest poor sleep quality 38 .

Cognitive variables
The Spanish version of the Pain Catastrophizing Scale (PCS), a PROM consisting of 13 items answered in a 5-point Likert scale (total scores range from 0 to 52), was used to assess catastrophizing responses (e.g., rumination, magnification, and despair aspects) 39 .
Fear of movement perceived by the patient was assessed with the Spanish version of the 11-items short-form of the Tampa Scale for Kinesiophobia (TSK-11) 40 .This PROM includes 11 items assessing the attitude of the patient against pain answered with a 4-point Likert scale (1: "complete disagreement" to 4: "complete agreement"), leading to a score ranging from 0 to 44 points.

Pressure pain thresholds
Pressure pain thresholds (PPTs) were bilaterally assessed at the upper trapezius, second metacarpal and tibialis anterior muscle with an electronic algometer (Somedic AB©, Farsta, Sweden) to identify the presence of widespread pressure pain sensitivity as a clinical manifestation of altered nociceptive pain processing.Pressure was applied at a rate of approximately 30 kPa/s on each point, three times on each location with a 30-s resting period between measurements.For analyses, the mean score of the three trials on each point was calculated.This testing procedure has shown good reliability (ICC ≥ 0.88) in patients with FMS 41 .

DNA collection and genotyping
Unstimulated whole saliva samples were always collected during the morning into collection tubes according to standardized procedures.Participants were asked not to eat, drink or chew gum for 1 h before sample collection.The saliva samples were collected, stored and centrifuged at 3000 rpm for 15 min no later than 48 h after collection to obtain the cell sediment and stored at -20º C until the analysis.This process is the same as used in previous studies 28,42 .
Genomic DNA was extracted from 500 ml of saliva using a MagMAX™ DNA Multi-Sample Ultra 2.0 Kit (Thermo Fisher Scientific Inc, Hemel Hempstead, Hertfordshire, UK) according to the manufacturer's protocol.We automatically extracted DNA using the King Fisher Flex purification robot (Thermo Fisher).The resulting DNA was assessed for purity and concentration using Quant-iT™ PicoGreen™ dsDNA reagent" (Thermo Fisher).The resulting DNA was diluted to 5 ng/μl, using 1 × Tris-EDTA (TE) buffer (Sigma-Aldrich, Dorset, UK).The qPCR reaction mixtures of 10ul contained a total of 10 ng gDNA as a PCR template, 1 × TaqMan Gene Expression PCR Master Mix and 0.6 × Genotyping TaqMan-probe assay.
For genotyping single nucleotide polymorphisms (SNP) with Real-Time PCR reaction TaqMan® Assay ID C_25746809_50 Predesigned SNP Genotyping Assays was used (Thermo Fisher Scientific Inc, Hemel Hempstead, Hertfordshire, UK).TaqMan® SNP Genotyping Assays use TaqMan® 5'-nuclease chemistry for amplifying and detecting specific alleles of each SNP in purified genomic DNA samples.Each assay allows the genotyping of individuals for a single nucleotide polymorphism.Real-time PCR plates were run in the Quantstudio 12 K Flex System (Thermo Fisher) of Genomics Unit (Madrid Science Park Foundation, Spain) under standard running conditions (95° for 10 min and 40 two-step cycles consisting of 95 °C for 15 s and 60 °C for 1 min) and analyzed with Genotyping App of Thermo Fisher Cloud.Identification of each of the three possible variants of each single nucleotide polymorphism was conducted by using specific fluorescent dyes.

Statistical analysis
Statistical analyses were performed using SPSS Statistics, version 25.0.Data were expressed as mean ± standard deviation (SD).The chi-squared (χ2) test was used to assess potential deviations from Hardy-Weinberg equilibrium.The Shapiro-Wilk test revealed that data exhibited normal distribution, hence, parametric tests were used.First, separate analyses of variance (ANOVA) were used to assess differences in clinical, functional, sensory-related, cognitive, and psychological variables according to the genotype of each polymorphism (OPRM1 rs1799971, HTR1B rs6296, COMT rs4680).Second, multilevel analysis of variance (MANOVA) including all polymorphisms (OPRM1 rs1799971, HTR1B rs6296, COMT rs4680) was also conducted to identify the possible interaction between all combinations of genotypes in the outcomes.Due to the inclusion of several variables, we applied correction by multiple testing considering a Bonferroni-corrected alpha of 0.0125 (4 time points).

Ethical approval
The study was conducted according to the guidelines of the Declaration of Helsinki and approved by the Clinical Ethics Committee of Universidad Rey Juan Carlos (URJC 08-30-2020).Written informed consent was obtained from all subjects involved in the study.

Results
From 140 Caucasian women with a diagnosis of FMS screened for eligible criteria, 14 (10%) were excluded due to previous major surgery (n = 8), previous whiplash (n = 4), and pregnancy (n = 2), leading to a final sample of 126 patients (mean age: 52 ± 9.9 years old).Three saliva samples were undetermined during genotyping analysis for the investigated SNPs and, accordingly, were excluded from the analysis providing a sample of 123 women with FMS with the complete data (Table 1).
The Asn/Asn genotype of the OPRM1 rs1799971 polymorphism was the most prevalent (63.4%), followed by the Asn/Asp (31.7%) and the Asp/Asp (4.9%) genotypes (Table 2).Thus, the Asn allele was present in 79.2% (n = 195/246) whereas the Asp allele was present in the remaining 20.8% (n = 51/246) of the sample.This genotype/allele distribution of the OPRM1 rs1799971 polymorphism did not deviate from that expected based on the Hardy-Weinberg equilibrium (chi square = 0.153).
The C/C genotype of the HTR1B rs6296 polymorphism was present in 51.2% (n = 63) of the sample, whereas the C/G and G/G genotypes were present in 41.4% (n = 51) and 7.4% (n = 9) of the sample (Table 3).Thus, the C allele was present in 71.9% (n = 177/246) whereas the G allele was present in the remaining 28.1% (n = 69/246) of the sample.This genotype/allele distribution of the HTR1B rs6296 polymorphism did not deviate from that expected based on the Hardy-Weinberg equilibrium (chi square = 0.091).
The Val/Val genotype of the COMT rs4680 polymorphism was present in 27.6% (n = 34) of the sample, whereas the Val/Met and Met/Met genotypes were present in 49.7% (n = 61) and 22.7% (n = 28) of the sample, respectively (Table 3).Thus, the Val allele was present in 52.4% (n = 129/246) whereas the Met allele was present in the remaining 47.6% (n = 117/246) of the sample.This genotype and allele distribution of the COMT rs4680 polymorphism deviated from that expected based on the Hardy-Weinberg equilibrium (chi square = 0.042).
Finally, the multivariate analysis did not reveal any statistical significant gene-to-gene interactions between OPRM1 rs1799971 x HTR1B rs6296, OPRM1 rs1799971 x COMT rs4680 and HTR1B rs6296 x COMT rs4680 for any of the investigated outcomes.

Discussion
This study examined the role of three polymorphisms, i.e., OPRM1 rs1799971, HTR1B rs6296 and COMT rs4680, commonly related to chronic pain within the clinical phenotype in women with FMS.Current results revealed that the OPRM1 rs1799971, HTR1B rs6296 and COMT rs4680 were not overall involved in phenotyping features of FMS related-pain.www.nature.com/scientificreports/

OPRM1 gene and fibromyalgia
Previous research has found that healthy people with the Asp allele exhibit lower pressure pain sensitivity compared with those carrying the Asn allele associated with the OPRM1 rs1799971 polymorphism 43 .This change is related to an alteration of the µ-opioid receptor, reducing or eliminating a putative site for N-linked glycosylation, leading to potentially altered interaction with β-endorphins 43 .In the current study, genotypes associated with the OPRM1 rs1799971 polymorphism did not show association with any of the investigated variables in our sample of women with FMS.Further, a previous study did not either find an association between OPRM1 rs1799971 polymorphism and a higher risk of developing FMS 15 .Current and previous findings would suggest that OPRM1 rs1799971 polymorphism genotypes are not related to pain in a specific chronic condition such as FMS.It is possible a potential effect of different or more than one type of opioid receptor, or a gene-to-gene interaction of this polymorphism in the clinical phenotype in women with FMS.

HTR1B gene and fibromyalgia
Similarly, we also observed that HTR1B rs6296 polymorphism, involved in the regulation of serotonin levels by inducing a synonymous mutation on the Val in position 287 18 , was not either associated with the clinical phenotype in women with FMS when investigated in isolation.Although this polymorphism has been associated with mental problems 44 , we did not either find significant differences in psychological or cognitive variables according to its genotypes.However, we do not know if other mental problems, such as suicide ideation 45 or the presence of post-traumatic stress disorder (PTSD) 46 , which are commonly seen in FMS, would be associated with these polymorphisms in this population.

COMT gene and fibromyalgia
The Val158Met rs4680 is the polymorphism most investigated in FMS; however, its association with a higher risk of suffering this condition is controversial [21][22][23][24] .The Met allele of this polymorphism results in an increased degradation of neurotransmitters such as dopamine, norepinephrine or epinephrine and, therefore, a reduction of these neurotransmitters at synaptic receptors 19 .We observed that women with FMS carrying the Met/Met genotype tend to report longer pain history, lower function and sensitization -associated symptoms than those www.nature.com/scientificreports/with the other genotypes; however, differences did not reach the statistical significance.Our results disagree with previous studies showing that patients with FMS and Met/Met carriers exhibit higher related-disability 25 and worse psychological aspects 28 .Similarly, an association with higher pain sensitivity, as a manifestation of central sensitization has been also associated with the Met/Met genotype in previous studies 26,27 .Nevertheless, it is important to consider that COMT variation has long been known to be more complex than the single Val/ Met polymorphism and studies looking at haplotypes of multiple SNPs are now necessary to account for this complexity.

Other polymorphisms and fibromyalgia
The fact that the investigated polymorphisms are not related to the pain phenotype or a higher risk of suffering a particular condition, in such a case FMS, does not exclude a genetic component in this pain condition.For instance, a comprehensive genome-wide investigation underscored that immediate family members are more susceptible to FMS, bolstering the assumption of a genetic foundation 47 .Potential genes accountable for pain susceptibility in FMS are the serotonin transporter gene (SLC64A4) and the transient receptor potential vanilloid 2 (TRPV2) gene 48 .In fact, SLC64A4, marked by a single nucleotide polymorphism, has been linked to chronic pain conditions such as temporo-mandibular pain as well as to elevated depression levels, and psychological disorders due to changes in serotonin reuptake 49 .Similarly, TRPV2 gene, which is expressed in mechano-and thermo-responsive neurons in the dorsal root and trigeminal ganglia, could be associated with the decreased pain thresholds observed in patients with FMS 11 .
It is also possible that other type of genetic analysis, such as DNA methylation, could reveal potential association between different genes and FMS.In fact, GRM2 methylation has revealed that this gene plays a relevant role in FMS patients since it causes an increase of cerebrospinal fluid glutamate levels 48 .Therefore, future studies investigating the association of these genes with the clinical phenotype in people with FMS are needed.www.nature.com/scientificreports/

Limitations
Finally, it is important to note some potential limitations to the current study.First, the sample only included women with FMS.Hence, extrapolation of these results to men with FMS should not be done.Therefore, investigating sex differences in future studies is needed.In addition, all participants were Caucasian, so we were not able to investigate the role of race or ethnicity in genetics.Lee et al. found that African-Americans were more likely to exhibit a Met/Met genotype than other ethnics, regardless of the diagnosis of FMS 24 .Thus, by excluding individuals with FMS taking analgesic drugs (aside from NSAIDs), we could unintentionally excluded patients with more pain and pain-related comorbidities.Accordingly, our results should be considered according to the features of our sample.Second, the sample size could be considered relatively small, and we did not conduct "a priori" sample size calculation leading to the identification of small samples (n = 6 or n = 9) in some allele homozygote genotypes which could be quite underpowered.Third, we did not include a control group; nevertheless, since we assessed the phenotype of a chronic pain condition, the inclusion of a pain-free control group would have not permitted assessment of these pain-related outcomes.Finally, we only assessed three polymorphisms associated with altered nociceptive processing.Future studies should investigate the influence of different polymorphisms, in addition to other genes in the clinical phenotype of this condition.

Conclusion
This study examined the role of three polymorphisms, i.e., OPRM1 rs1799971, HTR1B rs6296 and COMT rs4680, commonly related to chronic pain within the clinical phenotype in women with FMS.Current results revealed that the OPRM1 rs1799971, HTR1B rs6296 and COMT rs4680 were not overall involved in phenotyping features of FMS related-pain.Future studies including large sample sizes and a greater number of polymorphisms are needed. https://doi.org/10.1038/s41598-024-62240-7 https://doi.org/10.1038/s41598-024-62240-7www.nature.com/scientificreports/The possible variants of the OPRM1 rs1799971 polymorphism lead to the following genotypes: Asn/Asn (A/A), Asn/Asp (A/G) or Asp/Asp (G/G).The results were derived from a A → G substitution at the following sequence: GGT CAA CTT GTC CCA CTT AGA TGG C [A/G] ACC TGT CCG ACC CAT GCG GTC CGA A The variants of the HTR1B rs6296 polymorphism lead to the following genotypes: C/C, C/G or G/G.The results were derived from a C → G substitution at the sequence: CGG AGA CTC GCA CTT TGA CTT GGT T [C/G] ACA TAC ACA GGA GAT CCG GAT TCG C The possible variants of the COMT rs4680 polymorphism lead to the following three genotypes: Val/Val (G/G), Val/Met (G/A), Met/Met (A/A).The results were derived from A → G substitution at the following sequence: CCA GCG GAT GGT GGA TTT CGC TGG C [G/A] TGA AGG ACA AGG TGT GCA TGC CTG A. https://doi.org/10.1038/s41598-024-62240-7

Table 2 .
Differences in Clinical, Functional, Sensory-Related, Cognitive and Psychological variables as well as Pressure Pain Sensitivity in women with Fibromyalgia Syndrome Depending on the µ-Opioid Receptor Gene (OPRM1) Polymorphism (rs1799971).NPRS: Numerical Pain Rate Scale; FIQ: Fibromyalgia Impact Questionnaire; CSI: Central Sensitization Inventory; S-LANSS: self-reported version of the Leeds Assessment of Neuropathic Symptoms and Signs; PCS: Pain Catastrophizing Scale; TSK-11: Tampa Scale for Kinesiophobia; HADS: Hospital Anxiety and Depression Scale (A: Anxiety; D: Depression); PSQI: Pittsburgh Sleeping Quality Index; PPT: Pressure Pain Thresholds.

Table 4 .
Differences in Clinical, Functional, Sensory-Related, Cognitive and Psychological variables as well as Pressure Pain Sensitivity in women with Fibromyalgia Syndrome Depending on the Catechol-O-Methyltransferase Gene (COMT) Val158Met Polymorphism (rs4680).NPRS: Numerical Pain Rate Scale; FIQ: Fibromyalgia Impact Questionnaire; CSI: Central Sensitization Inventory; S-LANSS: self-reported version of the Leeds Assessment of Neuropathic Symptoms and Signs; PCS: Pain Catastrophizing Scale; TSK-11: Tampa Scale for Kinesiophobia; HADS: Hospital Anxiety and Depression Scale (A: Anxiety; D: Depression); PSQI: Pittsburgh Sleeping Quality Index; PPT: Pressure Pain Thresholds.

Table 1 .
Clinical, functional, sensory-related, cognitive, psychological, and psychophysical data of the sample.

Table 3 .
Differences in Clinical, Functional, Sensory-Related, Cognitive and Psychological variables as well as Pressure Pain Sensitivity in women with Fibromyalgia Syndrome Depending on the 5-hydroxytryptamine